Team:NTNU-Trondheim

Gram negative bacteria export numerous proteins into the periplasm. The twin-arginine translocation pathway (Tat pathway) is a protein export, or secretion pathway found in plants, bacteria, and archaea. The Tat pathway serves to actively translocate folded proteins across a lipid membrane bilayer. By using this transportationsystem we aim to introduce new proteins into the periplasm of bacteria. Once in the periplasm the protein will to some extent end up in outer membrane vesicles (OMV's) that budd of the bacteria.

As all gram negative bacteria produce outer membrane vesicles, we looked into the content of these vesicles. Was the sorting of proteins random? Could we direct certain proteins toward them? And what function would that give? Can we use this to our advantage?

Studies show that a twin-arginine signal peptide is able to direct the export of active green fluorescent protein (GFP) in E.coli and that translocation almost exclusively occur by the Tat-pathway. With this in mind we proceeded with making a construct containing tat and a GFP. As the goal of the project is to determine if vesicles can be utilized for drug delivery we want to see if they can be masked from the immunesystem by introducing a specific protein.

Protein G is known to bind to Human Serum Albumin which helps S.dysgalactiae subsp. equisimilis hide from the immune system. Protein G could therefore be a potential important piece in a drug carrier by masking it from immunological destruction. Introducing protein G into vesicles also demonstrate that it is indeed possible to manipulate the content and therefore the properties of OMV's.